Rett syndrome (RTT) is a devastating X-linked neurodevelopmental disorder and one of the leading causes of intellectual disability and developmental regression in girls. RTT is caused by loss-of-function mutations in the gene encoding the transcriptional modulator Methyl-CpG-Binding Protein 2 (MeCP2) and several mouse models that recapitulate features of the disease have been created by targeted disruption of the homologous mouse gene, Mecp2. There is a crucial need to develop therapies for RTT, however, the best practices and standards for performing preclinical trials ? which will be essential for prioritizing and validating therapies that are to be advanced to human trials ? have not yet been determined. To address this need, we propose studies in well-chosen RTT rodent models that consider factors such as sex, genetic strain background, species, and age during the natural course of disease. By defining the onset and progression of translationally-relevant neurobehavioral phenotypes and co-occurring plasma metabolite alterations, we will bridge behavioral outcome with potential biomarkers for RTT. In addition, we will use genetic and pharmacological strategies to examine how biomarkers may change with disease improvement to further classify markers that may predict treatment response. Finally, to optimize the clinical relevance of our results, we will test metabolites identified from our animal studies in RTT individuals. Our goal is to identify the phenotypic and biochemical alterations in Mecp2 rodents that can serve as outcome measures in preclinical studies in animal models and eventual clinical studies in humans. We hypothesize that abnormalities in plasma metabolites that co-occur with disease onset, become markedly altered with disease progression and conversely normalized with disease improvement will serve as the most useful biomarkers with the highest degree of translatability. The Specific Aims of the proposal are i) to define and validate translationally-relevant phenotypes and co-occurring changes in plasma metabolites among Mecp2 rodents, ii) to examine alterations in behavior and metabolite profile during disease improvement, and iii) to evaluate the predictive validity of Mecp2 rodent biochemical alterations in RTT. The unique features of the proposed work should maximize its utility to the RTT research community and accelerate preclinical studies in RTT models. Taken together, these studies will provide the indispensable ground-work for endeavors to identify from rodent models the interventions that have the highest likelihood of translating into effective human therapies. Regardless of the outcome, the results will define the direction that the field must take, either underscoring the need for new approaches, or promoting the most effective preclinical research practices using existing rodent models.